AB0026 CELL TYPE-SPECIFIC DYSREGULATION FOUND TO DIFFERENTIATE PATIENT SUBSETS OF SJÖGREN’S DISEASE
Authors: C. Li, B. Khatri, A. M. Stolarczyk, K. L. Tessneer, A. Rasmussen, J. Guthridge, J. A. James, R. H. Scofield, H. Li, A. D. Farris, K. Sivils, C. Lessard
Journal: Annals of the Rheumatic Diseases (2022)

Background Sjögren’s disease (SjD) is a chronic, heterogenous autoimmune disorder characterized by inflammatory destruction of the exocrine glands and Ro autoantibodies. Previous studies, most in whole blood, reported transcriptional and cell type differences between SjD subphenotypes. Objectives Evaluate the role of cell type-specific dysregulation in subpopulations of SjD patients having focal lymphocytic sialadenitis and/or anti-Ro/SSA and/or ANA positivity using single-cell RNA-seq (scRNA-seq). Methods Peripherical blood mononuclear cells (PBMCs) stored at -120°C from 30 SjD patients and 10 healthy controls (HCs) were selected from our OMRF Sjögren’s Research Cohort based on ACR/EULAR 2016 criteria. PBMCs were captured, libraries processed using 10X Genomics Chromium v3.1 and sequenced to a depth of ~45,000 reads/cell. 10X Genomics Cell Ranger (v 6.1.2) cell multiplexing pipeline was used to align reads to the GRCh38 human genome and separate combined data to per-sample output files. Seurat R package was used for quality control (QC), integration, clustering, and cell type assignment. Differential expression (DE) analysis was done using Seurat for genes with 10% or more of the cells showing expression. DE transcripts (defined as p adj &lt;0.05) were evaluated using Ingenuity Pathway Analysis (IPA). Results After removing cells with low feature counts, high mitochondrial gene counts, and/or other QC criteria, a total of 331,981 cells were captured. Cells were mapped to 28 clusters using Seurat, then cell types were predicted using marker genes developed from an in-house reference panel of publicly available scRNA-seq datasets. Mapping condensed the 28 observed cell clusters to 19 clusters of specific cell types; 4 clusters remained unidentified. Percentages of each cell type were compared across various subsets of SjD (Ro+; Ro-; Ro-/ANA+; Ro-/ANA-) and HCs. CD14 monocytes were decreased in Ro-/ANA- vs. Ro+ or Ro-/ANA+ cases (p&lt;0.05) and trended downward when compared to HCs (p=0.057). CD8 central memory T cells (TCM) were decreased in Ro+ vs. Ro- or Ro-/ANA- cases (p&lt;0.02) and trended downward when vs. HCs (p=0.076). Conventional dendric cells (DCs) were increased in Ro+ vs. Ro-/ANA- SjD cases (p=0.036) and trended upward in Ro+ vs. Ro- cases (p=0.08) but were not significantly different from HCs. Natural killer (NK) cells were increased in both Ro-/ANA- and Ro- vs. Ro+ (p&lt;0.05). DE analysis between immune cell subsets revealed potential subphenotype-specific differences in pathophysiology. Interferon signaling was upregulated in CD14 monocytes, naïve B, and CD4 central memory (TCM) cells from the Ro+ subset. Ro+ subset also showed upregulation of genes involved in T cell receptor signaling in CD4 cytotoxic T (CTL), CD4 TCM, and CD4 effector memory T (TEM) cells, but downregulation of this pathway in CD8 TCM cells. Ro+ subset also exhibited increased B cell receptor signaling pathway in naïve and memory B cells. Ro+ subset showed downregulation of autophagy in naïve B, CD4 CTL, CD4 TCM, and CD4 TEM cells. Several cell types in Ro+ and Ro-(ANA+/-) subsets showed decreased DE of genes involved in nitric oxide production and NRF2 oxidative stress pathways. Ro- (ANA+/-) vs. HCs showed increased IL6 and TREM1 signaling pathways in CD14 and CD16 monocytes. While Ro+ subsets showed upregulation of B and T cell pathways (shown previously in lupus), Ro- (ANA+/-) showed dysregulation of osteoarthritis and neuroinflammatory pathways. Interestingly, among the DE long intergenic non-coding (linc)RNAs found herein, we observed that LINC01871 , which we previously reported as DE in all subsets of SjD, was overexpressed in CD4 CTL and NK cells in both Ro+ and Ro- (ANA-) vs. HCs. Conclusion This interim analysis shows similarities and differences in the dysregulation of genes and pathways for specific cell subsets from our Ro+ and Ro- (ANA+/-) subsets of SjD, potentially allowing for more tailored diagnostics and interventions for the disease in the future. Disclosure of Interests Chuang Li: None declared, Bhuwan Khatri: None declared, Anna M Stolarczyk: None declared, Kandice L Tessneer: None declared, Astrid Rasmussen: None declared, Joel Guthridge: None declared, Judith A. James: None declared, R Hal Scofield: None declared, He Li Employee of: Current employee of Janssen, A Darise Farris Grant/research support from: Active collaborative research agreement with Janssen, Kathy Sivils Employee of: Current employee of Janssen, Christopher Lessard Grant/research support from: Active collaborative research agreement with Janssen